MLH1 methylation assay

ABSTRACT

The present technology relates to methods for excluding Lynch syndrome as a possible diagnosis in patients suffering from colorectal cancers or endometrial cancers. These methods are based on detecting the methylation status of the MLH1 promoter C region in colorectal and endometrial cancer patients using an improved and highly sensitive methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of PCT/US2016/028794,filed Apr. 22, 2016, which claims priority to U.S. ProvisionalApplication No. 62/151,744, filed Apr. 23, 2015, the contents of whichare hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 18, 2018, isnamed 034827-1238_SL.txt and is 4 KB in size.

TECHNICAL FIELD

The present technology relates to methods for excluding Lynch syndromeas a possible diagnosis in patients suffering from colorectal orendometrial cancers. These methods are based on detecting themethylation status of the MLH1 promoter in colorectal and endometrialcancer patients. Nucleic acid sequences that aid in the detection of themethylation status of the MLH1 promoter (such as primers and probes) arealso disclosed.

BACKGROUND

The following description of the background of the present technology isprovided simply as an aid in understanding the present technology and isnot admitted to describe or constitute prior art to the presenttechnology.

Alterations of DNA methylation patterns have been recognized as a commonchange in human cancers. Aberrant methylation of normally unmethylatedCpG-rich areas, also known as CpG-islands, which are located in or nearthe promoter region of many genes, have been associated withtranscriptional inactivation of important tumor suppressor genes, DNArepair genes, and metastasis inhibitor genes (Esteller, M. and Herman,J. G. J. Pathol. 196:1-7 (2002); Esteller, M. Lancet Oncol. 4:351-358(2003)). Therefore, detection of aberrant promoter methylation ofcancer-related genes may be essential for diagnosis, prognosis and/ordetection of metastatic potential of tumors. As the number of genesknown to be hypermethylated in cancer is large and increasing, sensitiveand robust multiplex methods for the detection of aberrant methylationof promoter regions are therefore desirable. In addition, the amount ofDNA available for large-scale studies is often limited and of poorquality because the DNA is isolated from formalin fixedparaffin-embedded (FFPE) tissues that have been stored at roomtemperature for years.

Most current approaches for the detection of methylation are based onthe conversion of unmethylated cytosine residues into uracil aftersodium bisulfite treatment (Frommer et al., Proc. Natl. Acad. Sci.89:1827-1831 (1992)), which are converted to thymidine during subsequentPCR. Thus, after bisulfite treatment, alleles that were originallymethylated have different DNA sequences as compared to theircorresponding unmethylated alleles. These differences can be exploitedby several techniques such as, methylation-specific PCR (MSP),restriction digestion (COBRA), Methylight, direct sequencing, denaturinghigh performance liquid chromatography (DHPLC), nucleotide extensionassays (MS-SnuPE), methylation-specific oligonucleotide (MSO)microarray, or HeavyMethyl (Frommer et al., supra; Cottrell et al.,Nucleic Acids Res. 32: e10 (2004); Deng et al., Nucleic Acids Res.30:E13 (2002); Eads et al., Nucleic Acids Res. 28, E32 (2000); Gitan etal., Genome Res. 12:158-164 (2002); Gonzalgo, M. & Jones, P. NucleicAcids Res. 25:2529-2531 (1997); Herman et al., Proc. Natl. Acad. Sci.93:9821-9826 (1996); Xiong, Z. & Laird, P., Nucleic Acids Res. 25,2532-2534 (1997)). However, most of these methods are labor intensiveand/or allow the study of the methylation status of only one gene at atime. In addition, most of these techniques are not suitable to studylarge numbers of paraffin-embedded tissue samples.

Commercially available Multiplex Ligation-dependent Probe Amplification(MLPA) kits are frequently used to detect methylation of the mismatchrepair (MMR) genes including MLH1. The MLPA method (U.S. Pub. No.2007/0092883) is based on the hybridization of hemi-probes to the targetDNA, each pair of which is separated by only one or a few bases. Eachhemi-probe is tagged with one of two universal sequences that are usedas priming sites for PCR amplification. Hybridization is followed byligation, and then amplification, using universal primers complementaryto the tags included at the end of each hemi-probe. Formethylation-specific MLPA (MS-MLPA), the ligation step is combined witha restriction endonuclease digestion step, using a methylation-sensitiveenzyme that cleaves unmethylated DNA at a specific site. Accordingly,any hemi-probe:target dimer in which the target DNA is unmethylated willbe digested, thereby failing to generate an intact sequence forexponential amplification.

Although the conventional MS-MLPA method obviates the need for bisulfiteconversion, MLPA kits are extremely sensitive to factors such asinhibitors present in the input DNA, operator differences, incubationtimes, etc., and thus reproducibility of these conventional MS-MLPAassays is often questionable. Thus, there is a substantial need for morerobust methods that effectively detect aberrant promoter methylation ofcancer-related genes.

SUMMARY OF THE PRESENT TECHNOLOGY

In one aspect, the present disclosure provides a method for detectingmethylation of a target nucleic acid sequence in the promoter of MLH1 ina sample comprising (a) incubating the sample comprising double-strandedgenomic DNA with at least one methylation-sensitive restriction enzymeand at least one methylation-insensitive restriction enzyme, wherein (i)the methylation-sensitive restriction enzyme and themethylation-insensitive restriction enzyme are not isoschizomers of eachother; (ii) the methylation-sensitive restriction enzyme cleaves thedouble-stranded genomic DNA at unmethylated recognition sites for themethylation-sensitive restriction enzyme, leaving methylated recognitionsites for the methylation-sensitive restriction enzyme intact; (iii) themethylation-insensitive restriction enzyme cleaves the double-strandedgenomic DNA at both methylated and unmethylated recognition sites forthe methylation-insensitive restriction enzyme; (iv) the target nucleicacid sequence in the promoter of MLH1 in the sample comprises arecognition site for the methylation-sensitive restriction enzyme; and(v) a target nucleic acid sequence at intron 14 of MLH1 in the samplecomprises a recognition site for the methylation-insensitive restrictionenzyme; (b) incubating the sample with a plurality of probes forquerying a plurality of target nucleic acids in the sample, wherein theplurality of probes comprises (i) a first locus specific probecomprising a first target specific region complementary to the targetnucleic acid sequence in the promoter of MLH1; and (ii) a second locusspecific probe comprising a second target specific region complementaryto the target nucleic acid sequence at intron 14 of MLH1, wherein thefirst locus specific probe and second locus specific probe aredetectably labelled; (c) hybridizing the plurality of probes to theplurality of target nucleic acids in the sample to form a plurality ofhybridization complexes; (d) amplifying the plurality of hybridizationcomplexes to produce a plurality of amplicons, wherein amplification iscarried out with a plurality of primer sets comprising (i) a firstforward primer comprising a region that is complementary to a nucleicacid sequence located 5′ from the target nucleic acid sequence in thepromoter of MLH1; (ii) a first reverse primer comprising a region thatis complementary to a nucleic acid sequence located 3′ from the targetnucleic acid sequence in the promoter of MLH1; (iii) a second forwardprimer comprising a region that is complementary to a nucleic acidsequence located 5′ from the target nucleic acid sequence at intron 14of MLH1; and (iv) a second reverse primer comprising a region that iscomplementary to a nucleic acid sequence located 3′ from the targetnucleic acid sequence at intron 14 of MLH1; and (e) detecting theplurality of amplicons, wherein detecting an amplicon comprising thetarget nucleic acid sequence in the promoter of MLH1 indicatesmethylation of the target nucleic acid sequence in the promoter of MLH1in the sample.

Additionally or alternatively, in some embodiments of the method, theplurality of probes further comprises a third locus specific probecomprising a third target specific region complementary to a targetnucleic acid sequence at ACTB, wherein the target nucleic acid sequenceat ACTB does not contain a recognition site for themethylation-sensitive restriction enzyme and the methylation-insensitiverestriction enzyme and wherein the third locus specific probe isdetectably labelled.

Additionally or alternatively, in some embodiments of the method, thefirst locus specific probe, the second locus specific probe and thethird locus specific probe are detectably labelled with fluorophores. Insome embodiments of the method, the fluorophores of the first locusspecific probe, the second locus specific probe, and the third locusspecific probe are distinct. In some embodiments of the method, thefluorophores are selected from the group consisting of FAM, CY5 and HEX.

In some embodiments of the method, the first locus specific probecomprises the sequence of 5′ AAGCACCTCCTCCGCTCTGC 3′ (SEQ ID NO: 1) or acomplement thereof. In some embodiments, the first locus specific probecomprises a 6-FAM fluorophore and a BHQ1 quencher moiety. Additionallyor alternatively, in some embodiments of the method, the second locusspecific probe comprises the sequence of 5′ CTACAACAATGGTCCAGGGAGCACA 3′(SEQ ID NO: 2) or a complement thereof. In some embodiments, the secondlocus specific probe comprises a HEX fluorophore and a BHQ1 quenchermoiety. In some embodiments of the method, the third locus specificprobe comprises the sequence of 5′ TGAACCTGTGTCTGCCACTGTGTG 3′ (SEQ IDNO: 3) or a complement thereof. In some embodiments, the third locusspecific probe comprises a Cy5 fluorophore and a BHQ2 quencher moiety.

Additionally or alternatively, in some embodiments of the method, theplurality of primer sets further comprises a third forward primercomprising a region that is complementary to a nucleic acid sequencelocated 5′ from the target nucleic acid sequence at ACTB; and a thirdreverse primer comprising a region that is complementary to a nucleicacid sequence located 3′ from the target nucleic acid sequence at ACTB.

In some embodiments of the method, the first forward primer comprisesthe sequence of 5′ AGGAGGAGCCTGAGAAGC 3′ (SEQ ID NO: 4) and the firstreverse primer comprises the sequence of 5′ CTTGTGTGCCTCTGCTGAG 3′ (SEQID NO: 5).

In some embodiments of the method, the second forward primer comprisesthe sequence of 5′ CTGAGTGTGTGAACAAGCAGAG 3′ (SEQ ID NO: 6) and thesecond reverse primer comprises the sequence of 5′ ACCTCATGCTGCTCTCCTTAG3′ (SEQ ID NO: 7).

In some embodiments of the method, the third forward primer comprisesthe sequence of 5′ GGCTCAGCAAGTCTTCTGG 3′ (SEQ ID NO: 8) and the thirdreverse primer comprises the sequence of 5′ CCTGGTGGGAAAGATGACC 3′ (SEQID NO: 9).

In some embodiments of the method, the methylation-sensitive restrictionenzyme is HhaI. In some embodiments of the method, themethylation-insensitive restriction enzyme is AluI.

In some embodiments of the method, the target nucleic acid sequence inthe promoter of MLH1 corresponds to the MLH1 promoter ‘C’ region.

In some embodiments of the method, the sample is a FFPE tissue sample.In some embodiments of the method, the sample is whole blood (WB).

In some embodiments of the method, the sample is derived from a subjectdiagnosed with colorectal or endometrial cancer. In one embodiment, thesubject diagnosed with colorectal or endometrial cancer is positive forthe BRAF V600E mutation. In another embodiment, the subject diagnosedwith colorectal or endometrial cancer is positive for microsatelliteinstability (MSI).

In some embodiments of the method, the sample is derived from a subjectsuspected of having Lynch syndrome. In some embodiments, the subjectsuspected of having Lynch syndrome displays tumors in one or moretissues selected from the group consisting of colon, rectum,endometrium, stomach, ovary, urinary tract, and small intestine. In oneembodiment, the tumors of the subject suspected of having Lynch syndromeshow a loss of MLH1 protein expression using immunohistochemical (IHC)methods. In other embodiments, the tumors of the subject suspected ofhaving Lynch syndrome are positive for MSI.

In another aspect, the present disclosure provides methods for excludingLynch syndrome as a possible diagnosis in a colorectal or endometrialcancer patient comprising interrogating the methylation status of theMLH1 promoter ‘C’ region in the colorectal or endometrial cancer patientusing the nucleic acids and methods described herein, whereinmethylation of the MLH1 promoter ‘C’ region indicates the absence ofLynch syndrome.

In some embodiments of the method, the patient displays tumors in one ormore tissues selected from the group consisting of colon, rectum,endometrium, stomach, ovary, urinary tract, and small intestine. In someembodiments of the method, the tumor tissue of the patient displays lossof MLH1 protein expression by immunohistochemistry (IHC). In someembodiments of the method, the tumor tissue of the patient is positivefor MSI. In some embodiments of the method, the tumor tissue of thepatient is positive for the BRAF V600E mutation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid sequences of the MLH1 promoter ‘C’ region,MLH1 intron 14, and ACTB amplicons. HhaI and AluI recognition sites arehighlighted in the relevant sequences. Primer sequences are depicted inuppercase font. Probe sequences are underlined. The primer/probelocations correspond to UCSC Genome Browser GRChv37/hg19 buildcoordinates. The three multiplex reactions (i.e., MLH1 promoter ‘C’region (SEQ ID NO: 10), MLH1 intron 14 (SEQ ID NO: 11), and ACTB (SEQ IDNO: 12)) are performed with an undigested no-enzyme control and aHhaI-AluI dual-digest for each sample.

FIG. 2A compares the real-time quantitative PCR data for the MLH1promoter ‘C’ region amplicon for samples digested with HhaI and AluIagainst undigested control samples. FIG. 2B compares the real-timequantitative PCR data for the MLH1 intron 14 amplicon for samplesdigested with HhaI and AluI against undigested control samples. FIG. 2Ccompares the real-time quantitative PCR data for the ACTB amplicon forsamples digested with HhaI and AluI against undigested control samples.Because the BRAF V600E mutation is commonly associated with MLH1promoter methylation in colorectal cancer (CRC) patients, V600E+ CRCpatient samples were tested as possible positives for MLH1 promotermethylation. MSI+ samples were also tested as possible positives forMLH1 promoter methylation because MSI can occur as a result of eitherMLH1 germline mutation or MLH1 promoter methylation. As used herein,“matched N of MSI-negative” and “matched N of BRAF V600E+” refer tomatched normal samples (e.g., normal tissue adjacent to the excisedtumor) of MSI-negative and V600E+ CRC patients respectively. As usedherein, “matched whole blood of MSI-negative” refers to a sample thatwas derived from a MSI-negative patient, where the matched normal sampleprovided along with the tumor tissue for screening was whole bloodinstead of adjacent normal tissue. As used herein, “whole blood from JAKvalidation” refers to a DNA sample that had been extracted from patientblood submitted for JAK2 testing. Both whole blood from JAK validationsamples and matched whole blood of MSI-negative samples serve aspossible negatives for MLH1 promoter methylation.

FIG. 3 is a Receiver Operating Characteristic (ROC) curve of the MLH1methylation assay of the present technology, which plots the assay'strue positive rate (sensitivity) against its false positive rate(100-Specificity) at different cutoff points for the “Methylation Score”(MeScore).

FIG. 4A shows a plot of the MeScore and corresponding bisulfiteconversion data for each tested sample (n=104). FIG. 4B shows thedetailed results of the MeScore and bisulfite conversion data for eachof the 104 samples depicted in FIG. 4A.

DETAILED DESCRIPTION

The present disclosure provides methods for excluding Lynch syndrome asa possible diagnosis in patients suffering from CRC or endometrialcancers. These methods are based on detecting the methylation status ofthe MLH1 promoter ‘C’ region in CRC and endometrial cancer patientsusing an improved and highly sensitive MS-MLPA assay. Nucleic acidsequences that aid in the detection of the methylation status of theMLH1 promoter ‘C’ region (such as primers and probes) are alsodisclosed.

DNA extracted from paraffin material is usually of poor quality and isnotoriously difficult to digest with restriction endonucleases. Storageof tissues in formaldehyde solution results in extensive crosslinking ofproteins to other proteins and to nucleic acids and in nucleic acidfragmentation (Grunau et al., Nucleic Acids Res. 29:E65 (2001); LehmannU. & Kreipe H., Methods 25:409-418 (2001)). Paraffin embedding is acommonly used technique, which results in partial denaturation of theDNA, making digestion of the sample DNA very difficult.

Previous studies (U.S. Pub. No. 2007/0092883) have demonstrated thatMS-MLPA methods involving the pre-digestion of genomic DNA with a CpGmethylation-sensitive restriction endonuclease, followed by denaturationand hybridization with MS-MLPA probes is accompanied by severaldrawbacks. Specifically, the salt conditions required for restrictionendonuclease digestion usually prevent the complete denaturation of thegenomic CpG islands by a simple heating step. Furthermore, these methodspreclude the analysis of most DNA samples derived from paraffin-embeddedtissue, most probably due to partial denaturation of DNA that isextracted from most paraffin-embedded tissues.

Conventional MS-MLPA kits circumvent the abovementioned drawbacks bycombining the ligation of MS-MLPA hemi-probes while hybridized to theirtarget sequence with simultaneous digestion of these MS-MLPAhemi-probe-DNA complexes with methylation-sensitive restrictionendonucleases. However, these kits do not account for inter-samplevariations in the cleavage activities of the methylation-sensitiverestriction endonucleases, making it difficult to determine whether alack of digestion is truly due to protection of the sequence bymethylation, or due to an inefficiency in the cleavage activity of themethylation-sensitive restriction enzyme within a given sample.Additionally, the performance of conventional MS-MLPA kits is impactedby a number of external factors such as inhibitors present in the inputDNA, operator differences, incubation times, etc., thereby impeding aninvestigator's ability to replicate results.

The present disclosure provides methods for detecting aberrantmethylation of the MLH1 promoter ‘C’ region using an improved and morerobust MS-MLPA assay that monitors the actual performance of themethylation-sensitive restriction endonuclease in each individual testsample. In particular, the methods disclosed herein include the use of amethylation-insensitive restriction enzyme as an internal control forenzymatic digestion in each test sample. The methods of the presenttechnology are useful in detecting MLH1 promoter methylation in genomicDNA derived from FFPE tissue samples, despite employing a step thatinvolves digesting the genomic DNA of the sample with multiplerestriction endonucleases. Accordingly, DNA degradation and partial DNAdenaturation during embedding of the tissues or longtime storage do notappear to influence the accuracy of the results of the MLH1 methylationassay of the present technology. Further, the methods of the presenttechnology are cost-effective and far less labor-intensive compared toother conventional methods for detecting methylation in genomic DNAsamples (e.g., methylation-specific real-time PCR, bisulfite conversion,MRC-Holland MLPA kits etc.).

Definitions

As used herein, the term “about” in reference to a number is generallytaken to include numbers that fall within a range of 1%, 5%, or 10% ineither direction (greater than or less than) of the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would be less than 0% or exceed 100% of a possible value).

As used herein, the terms “amplify” or “amplification” with respect tonucleic acid sequences, refer to methods that increase therepresentation of a population of nucleic acid sequences in a sample.Nucleic acid amplification methods, such as PCR, isothermal methods,rolling circle methods, etc., are well known to the skilled artisan.See, e.g., Saiki, “Amplification of Genomic DNA” in PCR PROTOCOLS, Inniset al., Eds., Academic Press, San Diego, Calif. 1990, pp 13-20; Wharamet al., Nucleic Acids Res. 2001 Jun. 1; 29(11):E54-E54; Hafner et al.,Biotechniques 2001 April; 30(4):852-6, 858, 860 passim. Copies of aparticular nucleic acid sequence generated in vitro in an amplificationreaction are called “amplicons” or “amplification products”.

The terms “complementary” or “complementarity” as used herein withreference to polynucleotides (i.e., a sequence of nucleotides such as anoligonucleotide or a target nucleic acid) refer to the base-pairingrules. The complement of a nucleic acid sequence as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.” For example, the sequence“5′-A-G-T-3′” is complementary to the sequence “3′-T-C-A-S.” Certainbases not commonly found in naturally-occurring nucleic acids may beincluded in the nucleic acids described herein. These include, forexample, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), andPeptide Nucleic Acids (PNA). Complementarity need not be perfect; stableduplexes may contain mismatched base pairs, degenerative, or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs. A complement sequence can also be anRNA sequence complementary to the DNA sequence or its complementsequence, and can also be a cDNA.

“Detecting” as used herein refers to determining the presence of amethylated nucleic acid of interest (e.g., MLH1 promoter ‘C’ region) ina sample. Detection does not require the method to provide 100%sensitivity.

“Detectable label” as used herein refers to a molecule or a compound ora group of molecules or a group of compounds used to identify a nucleicacid or protein of interest. In some embodiments, the detectable labelmay be detected directly. In other embodiments, the detectable label maybe a part of a binding pair, which can then be subsequently detected.Signals from the detectable label may be detected by various means andwill depend on the nature of the detectable label. Detectable labels maybe isotopes, fluorescent moieties, colored substances, and the like.Examples of means to detect detectable labels include but are notlimited to spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means, such as fluorescence,chemifluorescence, or chemiluminescence, or any other appropriate means.

The term “fluorophore” as used herein refers to a molecule that absorbslight at a particular wavelength (excitation frequency) and subsequentlyemits light of a longer wavelength (emission frequency). The term “donorfluorophore” as used herein means a fluorophore that, when in closeproximity to a quencher moiety, donates or transfers emission energy tothe quencher. As a result of donating energy to the quencher moiety, thedonor fluorophore will itself emit less light at a particular emissionfrequency than it would have in the absence of a closely positionedquencher moiety.

“Gene” as used herein refers to a DNA sequence that comprises regulatoryand coding sequences necessary for the production of an RNA, which mayhave a non-coding function (e.g., a ribosomal or transfer RNA) or whichmay include a polypeptide or a polypeptide precursor. The RNA orpolypeptide may be encoded by a full length coding sequence or by anyportion of the coding sequence so long as the desired activity orfunction is retained. Although a sequence of the nucleic acids may beshown in the form of DNA, a person of ordinary skill in the artrecognizes that the corresponding RNA sequence will have a similarsequence with the thymine being replaced by uracil, i.e., “T” isreplaced with “U.”

The term “hybridize” as used herein refers to a process where twosubstantially complementary nucleic acid strands (at least about 65%complementary over a stretch of at least 14 to 25 nucleotides, at leastabout 75%, or at least about 90% complementary) anneal to each otherunder appropriately stringent conditions to form a duplex orheteroduplex through formation of hydrogen bonds between complementarybase pairs. Hybridizations are typically and preferably conducted withprobe-length nucleic acid molecules, preferably 15-100 nucleotides inlength, more preferably 18-50 nucleotides in length. Nucleic acidhybridization techniques are well known in the art. See, e.g., Sambrook,et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Press, Plainview, N.Y. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is influenced by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, and the thermal melting point (T_(m)) of the formed hybrid.Those skilled in the art understand how to estimate and adjust thestringency of hybridization conditions such that sequences having atleast a desired level of complementarity will stably hybridize, whilethose having lower complementarity will not. For examples ofhybridization conditions and parameters, see, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994,Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus,N.J. In some embodiments, specific hybridization occurs under stringenthybridization conditions. An oligonucleotide or polynucleotide (e.g., aprobe or a primer) that is specific for a target nucleic acid will“hybridize” to the target nucleic acid under suitable conditions.

As used herein, the terms “individual”, “patient”, or “subject” can bean individual organism, a vertebrate, a mammal, or a human. In apreferred embodiment, the individual, patient or subject is a human.

As used herein, “isoschizomers” refer to pairs of restriction enzymesthat are specific to the same recognition sequence. Isoschizomers areisolated from different strains of bacteria and therefore may requiredifferent reaction conditions. In some embodiments, only one out of apair of isoschizomers can recognize both the methylated as well asunmethylated forms of recognition sites. In contrast, the otherrestriction enzyme can recognize only the unmethylated form of therecognition site. This property of some isoschizomers allowsidentification of the methylation status of the recognition site. Forexample, the restriction enzymes HpaII and MspI are isoschizomers, asthey both recognize the unmethylated form of the sequence 5′-CCGG-3′.However, only MspI can recognize the methylated form of the recognitionsite.

As used herein, the terms “+LR” or “positive likelihood ratio” refer tothe ratio between the probability of a positive test result given thepresence of a methylated MLH1 promoter ‘C’ region in a sample and theprobability of a positive test result given the absence of a methylatedMLH1 promoter ‘C’ region in a sample.

As used herein, the terms “-LR” or “negative likelihood ratio” refer tothe ratio between the probability of a negative test result given thepresence of a methylated MLH1 promoter ‘C’ region in a sample and theprobability of a negative test result given the absence of a methylatedMLH1 promoter ‘C’ region in a sample.

The term “MLH1 promoter” as used herein refers to a segment of the MLH1gene representing at least the first 250 nucleotides (nt) upstream fromthe translation start site of MLH1. In other embodiments, the promoterregion may include the first 250 nt, first 300 nt, first 350 nt, first400 nt, first 450 nt, first 500 nt, first 1 kb, first 5 kb, first 10 kb,first 15 kb, first 20 kb, first 21 kb or first 22 kb of sequencedirectly upstream of the start codon.

As used herein “MLH1 promoter ‘C’ region” refers to a small proximalregion of the MLH1 promoter comprising the nucleotides located atpositions 248 to 178 directly upstream from the translation start site.Methylation of the 8 CpG sites present in the MLH1 promoter ‘C’ regioncorrelate with the loss of MLH1 expression in CRC or endometrial cancer.

The term “multiplex PCR” as used herein refers to amplification of twoor more products which are each primed using a distinct primer pair.

As used herein, “oligonucleotide” refers to a molecule that has asequence of nucleic acid bases on a backbone comprised mainly ofidentical monomer units at defined intervals. The bases are arranged onthe backbone in such a way that they can bind with a nucleic acid havinga sequence of bases that are complementary to the bases of theoligonucleotide. The most common oligonucleotides have a backbone ofsugar phosphate units. A distinction may be made betweenoligodeoxyribonucleotides that do not have a hydroxyl group at the 2′position and oligoribonucleotides that have a hydroxyl group at the 2′position. Oligonucleotides may also include derivatives, in which thehydrogen of the hydroxyl group is replaced with organic groups, e.g., anallyl group. Oligonucleotides of the method which function as primers orprobes are generally at least about 10-15 nucleotides long and morepreferably at least about 15 to 25 nucleotides long, although shorter orlonger oligonucleotides may be used in the method. The exact size willdepend on many factors, which in turn depend on the ultimate function oruse of the oligonucleotide. The oligonucleotide may be generated in anymanner, including, for example, chemical synthesis, DNA replication,restriction endonuclease digestion of plasmids or phage DNA, reversetranscription, PCR, or a combination thereof. The oligonucleotide may bemodified e.g., by addition of a methyl group, a biotin or digoxigeninmoiety, a fluorescent tag or by using radioactive nucleotides.

As used herein, the term “primer” refers to an oligonucleotide, which iscapable of acting as a point of initiation of nucleic acid sequencesynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a target nucleic acid strandis induced, i.e., in the presence of different nucleotide triphosphatesand a polymerase in an appropriate buffer (“buffer” includes pH, ionicstrength, cofactors etc.) and at a suitable temperature. One or more ofthe nucleotides of the primer can be modified for instance by additionof a methyl group, a biotin or digoxigenin moiety, a fluorescent tag orby using radioactive nucleotides. A primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being substantially complementaryto the strand. The term primer as used herein includes all forms ofprimers that may be synthesized including peptide nucleic acid primers,locked nucleic acid primers, phosphorothioate modified primers, labeledprimers, and the like. The term “forward primer” as used herein means aprimer that anneals to the anti-sense strand of dsDNA. A “reverseprimer” anneals to the sense-strand of dsDNA.

“Probe” as used herein refers to nucleic acid that interacts with atarget nucleic acid via hybridization. A probe may be fullycomplementary to a target nucleic acid sequence or partiallycomplementary. The level of complementarity will depend on many factorsbased, in general, on the function of the probe. Probes can be labeledor unlabeled, or modified in any of a number of ways well known in theart. A probe may specifically hybridize to a target nucleic acid. Probesmay be DNA, RNA or a RNA/DNA hybrid. Probes may be oligonucleotides,artificial chromosomes, fragmented artificial chromosome, genomicnucleic acid, fragmented genomic nucleic acid, RNA, recombinant nucleicacid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA),locked nucleic acid, oligomer of cyclic heterocycles, or conjugates ofnucleic acid. Probes may comprise modified nucleobases, modified sugarmoieties, and modified internucleotide linkages. A probe may be used todetect the presence or absence of a methylated target nucleic acid.Probes are typically at least about 10, 15, 20, 25, 30, 35, 40, 50, 60,75, 100 nucleotides or more in length.

The term “quencher moiety” as used herein means a molecule that, inclose proximity to a donor fluorophore, takes up emission energygenerated by the donor and either dissipates the energy as heat or emitslight of a longer wavelength than the emission wavelength of the donor.In the latter case, the quencher is considered to be an acceptorfluorophore. The quenching moiety can act via proximal (i.e.,collisional) quenching or by Forster or fluorescence resonance energytransfer (“FRET”). Quenching by FRET is generally used in TaqMan® probeswhile proximal quenching is used in molecular beacon and Scorpion™ typeprobes.

As used herein, the “deltaCt value (dCt)” for a particular amplicon(e.g., MLH1 promoter ‘C’ region, MLH1 intron 14, or ACTB) refers to thedifference between the threshold cycle (Ct value) of the restrictionenzyme-digested sample (e.g., HhaI-AluI dual digested sample) and theundigested control sample.

As used herein, the term “Ratio C” represents the actual amount ofgenomic DNA digestion by the methylation-sensitive restrictionendonuclease (e.g., HhaI) normalized to the amount of total possiblegenomic DNA digestion in a given sample (which is measured by theactivity of a methylation-insensitive restriction endonuclease, such asAluI). Ratio C is calculated using the formula below:“Ratio C”=1−(MLH1 promoter ‘C’ region dCt/MLH1 intron 14 dCt)

Accordingly, a small difference in the dCt values for the MLH1 promoter‘C’ region and MLH1 intron 14 amplicons is indicative of completedigestion of the MLH1 promoter ‘C’ region, and thus reflects low levelsof methylation at the MLH1 promoter ‘C’ region. Conversely, a largedifference in the dCt values for the MLH1 promoter ‘C’ region and MLH1intron 14 amplicons is indicative of incomplete digestion of the MLH1promoter ‘C’ region, and thus reflects high levels of methylation at theMLH1 promoter ‘C’ region.

As used herein, the term “Ratio ACTB” represents the expected result for100% methylation at the MLH1 promoter ‘C’ region factoring in anyinter-well variability due to pipetting, etc. Ratio ACTB is calculatedusing the formula below:“Ratio ACTB”=1−(|ACTB dCt|/MLH1 intron 14 dCt)

The “MeScore” of a sample is computed using the formula below:“MeScore”=“Ratio C”/“Ratio ACTB”

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes each of which cleave double-strandedDNA at or near a specific nucleotide sequence known as a “restrictionsite”, “recognition site”, or “double-stranded recognition site.”

As used herein, a “sample” refers to a substance that is being assayedfor the presence of a methylated nucleic acid of interest (e.g., MLH1promoter). Processing methods to release or otherwise make available anucleic acid for detection are well known in the art and may includesteps of nucleic acid manipulation. A biological sample may be a bodyfluid or a tissue sample. In some cases, a biological sample may consistof or comprise blood, plasma, sera, urine, feces, epidermal sample,vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, culturedcells, bone marrow sample, tumor biopsies, and/or chorionic villi,cultured cells, and the like. Fixed or frozen tissues may also be used.Whole blood samples of about 0.5 to 5 ml collected with EDTA, ACD orheparin as anti-coagulant are suitable.

The term “sense strand” as used herein means the strand ofdouble-stranded DNA (dsDNA) that includes at least a portion of a codingsequence of a functional protein. “Anti-sense strand” means the strandof dsDNA that is the reverse complement of the sense strand.

The term “sensitivity” as used herein in reference to the methods of thepresent technology means the probability that a test result will bepositive when the MLH1 promoter ‘C’ region in a sample is methylated(true positive rate).

The term “specific” as used herein in reference to an oligonucleotideprimer means that the nucleotide sequence of the primer has at least 12bases of sequence identity with a portion of the nucleic acid to beamplified when the oligonucleotide and the nucleic acid are aligned. Anoligonucleotide primer that is specific for a nucleic acid is one that,under the stringent hybridization or washing conditions, is capable ofhybridizing to the target of interest and not substantially hybridizingto nucleic acids which are not of interest. Higher levels of sequenceidentity are preferred and include at least 75%, at least 80%, at least85%, at least 90%, at least 95% and more preferably at least 98%sequence identity.

The term “specificity” as used herein in reference to the methods of thepresent technology means the probability that a test result will benegative when the MLH1 promoter ‘C’ region in a sample is not methylated(true negative rate).

The term “stringent hybridization conditions” as used herein refers tohybridization conditions at least as stringent as the following:hybridization in 50% formamide, 5×SSC, 50 mM NaH₂PO₄, pH 6.8, 0.5% SDS,0.1 mg/mL sonicated salmon sperm DNA, and 5× Denhart's solution at 42°C. overnight; washing with 2×SSC, 0.1% SDS at 45° C.; and washing with0.2×SSC, 0.1% SDS at 45° C. In another example, stringent hybridizationconditions should not allow for hybridization of two nucleic acids whichdiffer over a stretch of 20 contiguous nucleotides by more than twobases.

The term “substantially complementary” as used herein means that twosequences hybridize under stringent hybridization conditions. Theskilled artisan will understand that substantially complementarysequences need not hybridize along their entire length. In particular,substantially complementary sequences may comprise a contiguous sequenceof bases that do not hybridize to a target sequence, positioned 3′ or 5′to a contiguous sequence of bases that hybridize under stringenthybridization conditions to a target sequence.

“TaqMan® PCR detection system” as used herein refers to a method forreal-time PCR. In this method, a TaqMan® probe which hybridizes to thenucleic acid segment amplified is included in the PCR reaction mix. TheTaqMan® probe comprises a donor and a quencher fluorophore on either endof the probe and in close enough proximity to each other so that thefluorescence of the donor is taken up by the quencher. However, when theprobe hybridizes to the amplified segment, the 5′-exonuclease activityof the Taq polymerase cleaves the probe thereby allowing the donorfluorophore to emit fluorescence which can be detected.

As used herein, the terms “target sequence” and “target nucleic acidsequence” refer to a specific nucleic acid sequence to be detectedand/or quantified in the sample to be analyzed.

CRC, Endometrial Cancer, and Lynch Syndrome

CRC is one of the most common malignancies, representing the third mostcommon cancer in men and the second in women worldwide. Endometrialcancer is the sixth most common cancer in women worldwide, with 320,000new cases diagnosed in 2012.

Microsatellites are repeated DNA sequences that occur approximatelyevery 50-100 Kb base pairs throughout the human genome. MSI is ahypermutable phenotype caused by the loss of DNA mismatch repairactivity and is implicated in the development of CRC and endometrialcancer. MSI is detected in about 15% of all CRCs; 3% are of which areassociated with Lynch syndrome and the other 12% are caused by sporadic,acquired hypermethylation of the promoter of the MLH1 gene, which occursin tumors with the CpG island methylator phenotype. Colorectal tumorswith MSI have distinctive features, including a tendency to arise in theproximal colon, lymphocytic infiltrate, and a poorly differentiated,mucinous or signet ring appearance. MSI is also present in endometrialcancer.

Hereditary nonpolyposis colon cancer (HNPCC), also known as Lynchsyndrome, is an inherited cancer syndrome caused by a germline mutationin one of several genes involved in DNA mismatch repair (MMR), includingMLH1, MSH2, MSH6 and PMS2. Lynch syndrome patients develop tumors atearly ages, often between 20 and 30 years old and frequently exhibitmultiple tumors, including those of the colon, rectum, endometrium,stomach, ovary, urinary tract, small intestine, and other sites, but noincrease in the frequency of cancers of the breast, lung, or prostate.

There are several laboratory-based strategies that help establish thediagnosis of Lynch syndrome, including testing tumor tissue for thepresence of MSI and loss of protein expression for any one of the MMRproteins by immunohistochemistry (IHC). However, the MSI tumor phenotypeis not restricted to inherited cancer cases; approximately 20% ofsporadic colon cancers are MSI. Thus, the presence of MSI does notdistinguish between a somatic (sporadic) and a germline (inherited)mutation, nor does it identify which gene is involved. IHC analysis,while helpful in identifying the affected gene, also does notdistinguish between somatic and germline defects.

Defective MMR in sporadic colon cancer is most often due to abnormalMLH1 promoter hypermethylation (epigenetic silencing). The region of theMLH1 promoter in which methylation mediates gene silencing is the 3′end, close to the start codon (e.g., the ‘C’ region). The 5′ end of thepromoter is also prone to methylation. Methylation of the 5′ end of theMLH1 promoter is not functionally relevant unless the methylationextends to the critical 3′ region. Therefore, specific CpG residues aremore important than others in mediating gene silencing. Importantly,most of the MSI-associated sporadic CRCs involve widespread CpG islandpromoter methylation (or CpG island methylator phenotype (CIMP)background), which is an important distinction from Lynch syndrometumors.

A specific mutation in the BRAF gene (V600E) has been shown to bepresent in approximately 70% of tumors with hypermethylation of the MLH1promoter. Importantly, the V600E mutation is rarely identified in caseswith germline MLH1 mutations (e.g., Lynch syndrome). Thus, directassessment of MLH1 promoter methylation status and testing for the BRAFV600E mutation are useful in distinguishing between a germline mutationand epigenetic/somatic inactivation of MLH1. Tumors that have the BRAFV600E mutation and demonstrate MLH1 promoter hypermethylation are almostcertainly sporadic, whereas tumors that show neither are most oftenassociated with an inherited disorder caused by a germline mutation(e.g., Lynch syndrome). The BRAF V600E mutation has been reported in CRCand endometrial cancers.

The likelihood of a germline mutation, e.g., a mutation present in Lynchsyndrome, is very low in situations where the tumor demonstrates MLH1promoter hypermethylation and the normal tissue is unmethylated. Thelikelihood of a germline mutation is high in those cases where the tumorand normal tissue lack MLH1 promoter hypermethylation.

Real-Time Quantitative PCR

Amplification of target nucleic acids can be detected by any of a numberof methods well-known in the art such as gel electrophoresis, columnchromatography, hybridization with a probe, sequencing, melting curveanalysis, or “real-time” detection.

For real-time detection, primers and/or probes may be detectably labeledto allow differences in fluorescence when the primers becomeincorporated or when the probes are hybridized, for example, andamplified in an instrument capable of monitoring the change influorescence during the reaction. Real-time detection methods fornucleic acid amplification are well known and include, for example, theTaqMan® system, Scorpion™ primer system and use of intercalating dyesfor double-stranded nucleic acids.

In real-time quantitative PCR, the accumulation of amplification productis measured continuously in both standard dilutions of target DNA andsamples containing unknown amounts of target DNA. A standard curve isconstructed by correlating initial template concentration in thestandard samples with the number of PCR™ cycles (Ct) necessary toproduce a specific threshold concentration of product. In the testsamples, target PCR™ product accumulation is measured after the same Ct,which allows interpolation of target DNA concentration from the standardcurve.

In some embodiments, amplified nucleic acids are detected byhybridization with a specific probe. Probe oligonucleotides,complementary to a portion of the amplified target sequence may be usedto detect amplified fragments. In some embodiments, hybridization may bedetected in real time. Amplified nucleic acids for each of the targetsequences may be detected simultaneously (i.e., in the same reactionvessel) or individually (i.e., in separate reaction vessels). Forsequence-modified nucleic acids, the target may be independentlyselected from the top strand or the bottom strand. Thus, all targets tobe detected may comprise top strand, bottom strand, or a combination oftop strand and bottom strand targets.

One general method for real-time PCR uses fluorescent probes such as theTaqMan® probes, molecular beacons, and Scorpions. Real-time PCRquantifies the initial amount of the template with more specificity,sensitivity and reproducibility, than other forms of quantitative PCR,which detect the amount of final amplified product. Real-time PCR doesnot detect the size of the amplicon. The probes employed in Scorpion™and TaqMan® technologies are based on the principle of fluorescencequenching and involve a donor fluorophore and a quenching moiety.

Real time PCR is performed using any suitable instrument capable ofdetecting the accumulation of the PCR amplification product. Mostcommonly, the instrument is capable of detecting fluorescence from oneor more fluorescent labels. For example, real time detection on theinstrument (e.g., an ABI Real-Time PCR System 7500® sequence detector)monitors fluorescence and calculates the measure of reporter signal, orRn value, during each PCR cycle. The threshold cycle, or Ct value, isthe cycle at which fluorescence intersects the threshold value. Thethreshold value can be determined by the sequence detection systemsoftware or manually.

TaqMan® probes (Heid et al., Genome Res. 6: 986-994, 1996) use thefluorogenic 5′ exonuclease activity of Taq polymerase to measure theamount of target sequences in DNA samples. TaqMan® probes areoligonucleotides that contain a donor fluorophore usually at or near the5′ base, and a quenching moiety typically at or near the 3′ base. Thequencher moiety may be a dye such as TAMRA or may be a non-fluorescentmolecule such as 4-(4-dimethylaminophenylazo) benzoic acid (DABCYL). SeeTyagi et al., 16 Nature Biotechnology 49-53 (1998). When irradiated, theexcited fluorescent donor transfers energy to the nearby quenchingmoiety by FRET rather than fluorescing. Thus, the close proximity of thedonor and quencher prevents emission of donor fluorescence while theprobe is intact.

TaqMan® probes are designed to anneal to an internal region of a PCRproduct. When the polymerase replicates a template on which a TaqMan®probe is bound, its 5′ exonuclease activity cleaves the probe. Thisterminates the activity of the quencher (no FRET) and the donorfluorophore starts to emit fluorescence which increases in each cycleproportional to the rate of probe cleavage. Accumulation of PCR productis detected by monitoring the increase in fluorescence of the reporterdye. If the quencher is an acceptor fluorophore, then accumulation ofPCR product can be detected by monitoring the decrease in fluorescenceof the acceptor fluorophore.

In some embodiments, the detectable label is a fluorophore. Suitablefluorescent moieties include but are not limited to the followingfluorophores working individually or in combination:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate; Alexa Fluors: AlexaFluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, AlexaFluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes);5-(2-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BlackHole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes:BODIPY® R-6G, BOPIPY® 530/550, BODIPY® FL; Brilliant Yellow; coumarinand derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®,Cy5®, Cy5.5®; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); Eclipse™(Epoch Biosciences Inc.); eosin and derivatives: eosin, eosinisothiocyanate; erythrosin and derivatives: erythrosin B, erythrosinisothiocyanate; ethidium; fluorescein and derivatives:5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein(JOE), fluorescein, fluorescein isothiocyanate (FITC),hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC),tetrachlorofluorescem (TET); fluorescamine; IR144; IR1446; lanthamidephosphors; Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde;Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrenebutyrate, succinimidyl 1-pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY®35 (Molecular Probes); Reactive Red 4 (Cibacron® Brilliant Red 3B-A);rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamineX isothiocyanate, riboflavin, rosolic acid, sulforhodamine B,sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101(Texas Red); terbium chelate derivatives;N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); and VIC®.

Suitable quenchers are selected based on the fluorescence spectrum ofthe particular fluorophore. Useful quenchers include, for example, theBlack Hole™ quenchers BHQ-1, BHQ 2, and BHQ-3 (Biosearch Technologies,Inc.), and the ATTO-series of quenchers (ATTO 540Q, ATTO 580Q, and ATTO612Q; Atto-Tec GmbH).

MLH1 Methylation Assay of the Present Technology

The methods of the present technology are based on the principle thatthe target nucleic acid sequence in the promoter of MLH1 contains arestriction site recognized by a methylation-sensitive restrictionendonuclease, such as HhaI, that is sensitive to cytosine methylation ofat least one CpG site in its recognition sequence. Upon digestion with amethylation-sensitive restriction endonuclease, an amplification productof the target nucleic acid sequence in the promoter of MLH1 will only beobtained if the CpG site is methylated. The amplification product of thetarget nucleic acid sequence in the promoter of MLH1 is detected viareal-time PCR.

It is to be understood that any methylated site present in a targetnucleic acid in the promoter of MLH1 can be detected by the methodsdisclosed herein, as long as the site of interest (as part of adouble-stranded recognition sequence) can be recognized by amethylation-sensitive restriction endonuclease (e.g., HhaI), cleavingthe nucleic acid when the sequence of interest is unmethylated, andleaving the nucleic acid uncleaved when the sequence is methylated.

In addition to detecting the presence of a methylated target nucleicacid sequence in the promoter of MLH1 in a sample viamethylation-sensitive restriction enzyme digestion in combination withreal-time quantitative PCR, the assay evaluates the digestion of analternate region of the MLH1 gene (e.g., at intron 14) by amethylation-insensitive restriction enzyme that is not an isoschizomerof the methylation-sensitive restriction enzyme (e.g.; AluI), as aninternal control for complete enzymatic digestion within the sample.

Thus, the methods of the present technology account for variations inthe cleavage activities of the methylation-sensitive restrictionendonucleases from one sample to another, thereby allowing aninvestigator to discriminate between the lack of digestion being due toprotection of the sequence by methylation, or due to an inefficiency inthe cleavage activity of the restriction enzyme.

In some embodiments of the method, a sample comprising a controlunmethylated target nucleic acid is provided to ensure the properactivity of the methylation-sensitive restriction enzyme andmethylation-insensitive restriction enzyme.

In some embodiments of the method, a control sample lacking both themethylation-sensitive restriction enzyme and methylation-insensitiverestriction enzyme is included in order to check the maximum performanceof the system. In such situations, all the target nucleic acids,regardless of their methylation status, will be amplified.

In some embodiments, a sample comprising genomic DNA is split into twoaliquots, one of which is simultaneously digested with both amethylation-sensitive restriction enzyme and a methylation-insensitiverestriction enzyme, while the other is incubated in digestion bufferlacking either restriction enzyme.

In some embodiments of the method, amplification of a region (e.g.,ACTB) that lacks restriction sites for both the methylation-sensitiverestriction enzyme and methylation-insensitive restriction enzyme isincluded as an internal control for the complete absence of enzymaticdigestion.

In some embodiments of the method, the level of methylation of a targetnucleic acid sequence in the promoter of MLH1 in a sample is quantifiedby computing the MeScore as defined herein.

Thus according to the methods of the present technology, if the MLH1promoter ‘C’ region is methylated (and thus protected from digestion bythe methylation-sensitive restriction endonuclease), the MLH1 promoter‘C’ region amplicon will be detected, and yield a Ct value that iscomparable to that observed in the corresponding undigested controlsample that lacks both the methylation-sensitive restriction enzyme andmethylation-insensitive restriction enzyme.

However, if the targeted CpG sites in the MLH1 promoter ‘C’ region areunmethylated, the MLH1 promoter ‘C’ region will be cleaved at or nearthe recognition sites for the methylation-sensitive restrictionendonuclease, thereby reducing the amount of intact target nucleic acidsequence available for the amplification and detection of the MLH1promoter ‘C’ region amplicon. In light of the high sensitivity ofreal-time quantitative PCR assays, it is understood that anyunmethylated DNA left intact after being subjected tomethylation-sensitive restriction endonuclease digestion will beamplified and detected. Thus the resulting MLH1 promoter ‘C’ regionamplicon would yield a higher Ct value relative to that observed in thecorresponding undigested control sample that lacks both themethylation-sensitive restriction enzyme and methylation-insensitiverestriction enzyme.

A heterogeneous positive sample containing a large fraction of normalDNA mixed with tumor DNA, and therefore a mixture of methylated andunmethylated DNA in the MLH1 promoter ‘C’ region, would thus undergo anintermediate level of digestion at the targeted CpG sites, and yield anintermediate upward shift in Ct value.

The methods disclosed herein require that the Ct values for the MLH1promoter ‘C’ region amplicon in a given sample be compared to thecorresponding Ct values for the MLH1 intron 14 amplicon (see below) inthe same sample to confirm that the detected signal for the MLH1promoter ‘C’ region is due to protection of the sequence by methylation(a true positive), rather than an inefficiency in the cleavage activityof the methylation-sensitive restriction enzyme within a sample. Forexample, the presence of carryover of inhibitory agents in the input DNAof a methylation-negative sample may result in a smaller-than-averageshift in Ct value for the MLH1 promoter ‘C’ region amplicon, which wouldsuggest the presence of methylation (a false-positive call). Similarly,a high degree of genomic DNA fragmentation in a sample would also leadto false positive calls. The methods disclosed herein overcome thesedrawbacks by assaying the enzymatic activity of amethylation-insensitive restriction endonuclease as an internalreference for enzymatic digestion within the sample.

The MLH1 intron 14 region (located at the exon-14-intron 14 junction)contains a restriction site recognized by the methylation-insensitiverestriction enzyme (e.g., AluI). The MLH1 intron 14 region in a samplewill be cleaved at or near the recognition sites for themethylation-insensitive restriction endonuclease, regardless of themethylation status of the region. Digestion of the MLH1 intron 14 regionthus reduces the amount of intact target nucleic acid sequence availablefor the amplification and detection of the MLH1 intron 14 amplicon.

Any intact target nucleic acid sequence in the MLH1 intron 14 regionthat persists after digestion with the methylation-insensitiverestriction enzyme will be amplified and detected. Thus the resultingMLH1 intron 14 amplicon would yield a higher Ct value relative to thatobserved in the corresponding undigested control sample that lacks boththe methylation-sensitive restriction enzyme and methylation-insensitiverestriction enzyme.

In some embodiments of the method, amplification of a region (e.g.,ACTB) that lacks restriction sites for both the methylation-sensitiverestriction enzyme and methylation-insensitive restriction enzyme isincluded as an internal control for the complete absence of enzymaticdigestion. Thus, the ACTB amplicon in the sample digested with both themethylation-sensitive restriction enzyme and methylation-insensitiverestriction enzyme will yield a Ct value that is nearly identical tothat observed in the corresponding undigested control sample that lacksboth the methylation-sensitive restriction enzyme andmethylation-insensitive restriction enzyme. In some embodiments of themethod, the deltaCt value for the ACTB amplicons between the undigestedand digested samples accounts for any intra-sample variability, such asthose created by pipetting errors, etc.

In some embodiments of the method, the deltaCt value for the MLH1promoter ‘C’ region amplicon in a given sample is compared to thecorresponding deltaCt value for the ACTB amplicon in the same sample,wherein a low difference in deltaCt values between the MLH1 promoter ‘C’region amplicon and the ACTB amplicon is indicative of high levels ofmethylation.

In another embodiment, a plurality of different methylation-sensitiverestriction enzymes can be used in a single or in separate reactions todetect multiple methylated sites within a single or a plurality oftarget nucleic acid sequences in the promoter of MLH1 in a sample,wherein the different methylation-sensitive restriction enzymes are notisoschizomers of each other.

In another aspect, the present disclosure provides robust methods fordetecting aberrant methylation of the MLH1 promoter in DNA samplesextracted from FFPE tissue.

Identification of the Risk of Lynch Syndrome in Patients Suffering fromCRC or Endometrial Cancer

The methods disclosed herein can provide useful diagnostic informationwhen evaluating a patient suspected of having Lynch syndrome, especiallywhen testing is performed in conjunction with HNPCC/HereditaryNonpolyposis Colorectal Cancer (HNPCC) Screen, which includes MSI andIHC studies.

The described methods for detecting the presence of aberrant methylationof the MLH1 promoter in a sample may be used for determining whether apatient suffering from CRC or endometrial cancer should be diagnosedwith Lynch syndrome. In some embodiments of the method, the tumor tissueof the patient displays loss of MLH1 protein expression byimmunohistochemistry (IHC). In some embodiments of the method, the tumortissue of the patient is positive for MSI. In some embodiments of themethod, the tumor tissue of the patient is positive for the BRAF V600Emutation.

In one embodiment, the present technology provides a method forexcluding Lynch syndrome as a possible diagnosis in a CRC or endometrialcancer patient comprising (a) incubating a double-stranded genomic DNAsample obtained from the patient with at least one methylation-sensitiverestriction enzyme and at least one methylation-insensitive restrictionenzyme, wherein (i) the methylation-sensitive restriction enzyme and themethylation-insensitive restriction enzyme are not isoschizomers of eachother; (ii) the methylation-sensitive restriction enzyme cleaves thedouble-stranded genomic DNA at unmethylated recognition sites for themethylation-sensitive restriction enzyme, leaving methylated recognitionsites for the methylation-sensitive restriction enzyme intact; (iii) themethylation-insensitive restriction enzyme cleaves the double-strandedgenomic DNA at both methylated and unmethylated recognition sites forthe methylation-insensitive restriction enzyme; (iv) a target nucleicacid sequence in the promoter of in the sample comprises a recognitionsite for the methylation-sensitive restriction enzyme; and (v) a targetnucleic acid sequence at intron 14 of MLH1 in the sample comprises arecognition site for the methylation-insensitive restriction enzyme; (b)incubating the sample with a plurality of probes for querying aplurality of target nucleic acids in the sample, wherein the pluralityof probes comprises (i) a first locus specific probe comprising a firsttarget specific region complementary to the target nucleic acid sequencein the promoter of MLH1; and (ii) a second locus specific probecomprising a second target specific region complementary to the targetnucleic acid sequence at intron 14 of MLH1, wherein the first locusspecific probe and second locus specific probe are detectably labelled;(c) hybridizing the plurality of probes to the plurality of targetnucleic acids in the sample to form a plurality of hybridizationcomplexes; (d) amplifying the plurality of hybridization complexes toproduce a plurality of amplicons, wherein amplification is carried outwith a plurality of primer sets comprising (i) a first forward primercomprising a region that is complementary to a nucleic acid sequencelocated 5′ from the target nucleic acid sequence in the promoter ofMLH1; (ii) a first reverse primer comprising a region that iscomplementary to a nucleic acid sequence located 3′ from the targetnucleic acid sequence in the promoter of MLH1; (iii) a second forwardprimer comprising a region that is complementary to a nucleic acidsequence located 5′ from the target nucleic acid sequence at intron 14MLH1; and (iv) a second reverse primer comprising a region that iscomplementary to a nucleic acid sequence located 3′ from the targetnucleic acid sequence at intron 14 of MLH1; and (e) detecting theplurality of amplicons, wherein detecting an amplicon comprising thetarget nucleic acid sequence in the promoter of MLH1 indicatesmethylation of the target nucleic acid sequence in the promoter of MLH1in the sample and the absence of Lynch syndrome in the patient.

Additionally or alternatively, in some embodiments of the method, theplurality of probes further comprises a third locus specific probecomprising a third target specific region complementary to a targetnucleic acid sequence at ACTB, wherein the target nucleic acid sequenceat ACTB does not contain a recognition site for themethylation-sensitive restriction enzyme and the methylation-insensitiverestriction enzyme and wherein the third locus specific probe isdetectably labelled.

Additionally or alternatively, in some embodiments of the method, thefirst locus specific probe, the second locus specific probe and thethird locus specific probe are detectably labelled with fluorophores. Insome embodiments of the method, the first locus specific probe, thesecond locus specific probe, and the third locus specific probe arelabelled with a distinct fluorophore to allow discrimination between thedetected amplicons. In some embodiments of the method, the fluorophoresare selected from the group consisting of FAM, CY5 and HEX.

In some embodiments of the method, the first locus specific probecomprises the sequence of 5′ AAGCACCTCCTCCGCTCTGC 3′ (SEQ ID NO: 1) or acomplement thereof. In some embodiments, the first locus specific probecomprises a 6-FAM fluorophore and a BHQ1 quencher moiety. Additionallyor alternatively, in some embodiments of the method, the second locusspecific probe comprises the sequence of 5′ CTACAACAATGGTCCAGGGAGCACA 3′(SEQ ID NO: 2) or a complement thereof. In some embodiments, the secondlocus specific probe comprises a HEX fluorophore and a BHQ1 quenchermoiety. In some embodiments of the method, the third locus specificprobe comprises the sequence of 5′ TGAACCTGTGTCTGCCACTGTGTG 3′ (SEQ IDNO: 3) or a complement thereof. In some embodiments, the third locusspecific probe comprises a Cy5 fluorophore and a BHQ2 quencher moiety.

Additionally or alternatively, in some embodiments of the method, theplurality of primer sets further comprises a third forward primercomprising a region that is complementary to a nucleic acid sequencelocated 5′ from the target nucleic acid sequence at ACTB; and a thirdreverse primer comprising a region that is complementary to a nucleicacid sequence located 3′ from the target nucleic acid sequence at ACTB.

In some embodiments of the method, the first forward primer comprisesthe sequence of 5′ AGGAGGAGCCTGAGAAGC 3′ (SEQ ID NO: 4) and the firstreverse primer comprises the sequence of 5′ CTTGTGTGCCTCTGCTGAG 3′ (SEQID NO: 5).

In some embodiments of the method, the second forward primer comprisesthe sequence of 5′ CTGAGTGTGTGAACAAGCAGAG 3′ (SEQ ID NO: 6) and thesecond reverse primer comprises the sequence of 5′ ACCTCATGCTGCTCTCCTTAG3′ (SEQ ID NO: 7).

In some embodiments of the method, the third forward primer comprisesthe sequence of 5′ GGCTCAGCAAGTCTTCTGG 3′ (SEQ ID NO: 8) and the thirdreverse primer comprises the sequence of 5′ CCTGGTGGGAAAGATGACC 3′ (SEQID NO: 9).

In some embodiments of the method, the methylation-sensitive restrictionenzyme is HhaI. In some embodiments of the method, themethylation-insensitive restriction enzyme is AluI.

In some embodiments of the method, the target nucleic acid sequence inthe promoter of MLH1 corresponds to the MLH1 promoter ‘C’ region.

In some embodiments of the method, the sample is a FFPE tissue sample.In some embodiments of the method, the sample is WB.

The methods disclosed herein can also be used to determine whether apatient suffering from CRC or endometrial cancer is a suitable candidatefor Lynch syndrome therapies. Lynch syndrome therapies includecolectomy, oophorectomy and hysterectomy.

In certain embodiments, the present disclosure provides methods fordetermining whether a patient suffering from CRC or endometrial canceris a suitable candidate for Lynch syndrome therapies comprisinginterrogating the methylation status of the MLH1 promoter ‘C’ region inthe CRC or endometrial cancer patient using the nucleic acids andmethods described herein, wherein methylation of the MLH1 promoter ‘C’region indicates that the patient is not a suitable candidate for Lynchsyndrome therapies.

Kits

The present disclosure also provides kits for detecting the methylationstatus of the MLH1 promoter via the improved MS-MLPA methods disclosedherein. Kits of the present technology comprise one or moretarget-specific nucleic acid probes as disclosed herein (e.g., probesspecific to MLH1 promoter C region, MLH1 intron 14 or ACTB), alone or incombination with one or more primer pairs as disclosed herein, foramplification and detection of methylated target nucleic acid sequenceswithin the genomic DNA of a given sample.

In some embodiments, the kits provide a target-specific nucleic acidprobe comprising at least a part of a single stranded sequenceconstituting one of the strands of a double stranded recognition site ofa methylation-sensitive restriction enzyme. In some embodiments, thetarget-specific nucleic acid probe comprising at least a part of asingle stranded sequence constituting one of the strands of a doublestranded recognition site of a methylation-sensitive restriction enzymecomprises the sequence 5′ AAGCACCTCCTCCGCTCTGC 3′ (SEQ ID NO: 1) or acomplement thereof.

In some embodiments, the kits provide a target-specific nucleic acidprobe comprising at least a part of a single stranded sequenceconstituting one of the strands of a double stranded recognition site ofa methylation-insensitive restriction enzyme. In some embodiments, thetarget-specific nucleic acid probe comprising at least a part of asingle stranded sequence constituting one of the strands of a doublestranded recognition site of a methylation-insensitive restrictionenzyme comprises the sequence 5′ CTACAACAATGGTCCAGGGAGCACA 3′ (SEQ IDNO: 2) or a complement thereof.

In some embodiments, the kits provide a target-specific nucleic acidprobe comprising a single stranded sequence lacking a recognition sitefor both a methylation-insensitive restriction enzyme and amethylation-sensitive restriction enzyme. In some embodiments, thetarget-specific nucleic acid probe comprising a single stranded sequencelacking a recognition site for both a methylation-insensitiverestriction enzyme and a methylation-sensitive restriction enzymecomprises the sequence 5′ TGAACCTGTGTCTGCCACTGTGTG 3′ (SEQ ID NO: 3) ora complement thereof.

In some embodiments, the kit comprises a mixture of target nucleic acidprobes which comprises at least a first target nucleic acid probe and asecond target nucleic acid probe and optionally a third target nucleicacid probe, wherein at least one of the probes comprises at least a partof a single stranded sequence, constituting one of the strands of adouble stranded recognition site of a methylation-sensitive restrictionenzyme.

Additionally or alternatively, the kits comprise one or more primerpairs selected from the group consisting of 5′ AGGAGGAGCCTGAGAAGC 3′(SEQ ID NO: 4) and 5′ CTTGTGTGCCTCTGCTGAG 3′ (SEQ ID NO: 5); 5′CTGAGTGTGTGAACAAGCAGAG 3′ (SEQ ID NO: 6) and 5′ ACCTCATGCTGCTCTCCTTAG 3′(SEQ ID NO: 7); and 5′ GGCTCAGCAAGTCTTCTGG 3′ (SEQ ID NO: 8) and 5′CCTGGTGGGAAAGATGACC 3′ (SEQ ID NO: 9).

In some embodiments, the kit comprises liquid medium containing the atleast one target-specific nucleic acid probe in a concentration of 250nM or less. With such a kit, the probes are provided in the requiredamount to perform reliable multiplex detection reactions according tothe present technology. In some embodiments, the target-specific nucleicacid probes are detectably labeled.

In some embodiments, the kits further comprise buffers,methylation-sensitive and methylation-insensitive restrictionendonucleases, enzymes having polymerase activity, enzymes havingpolymerase activity and lacking 5→3′ exonuclease activity or both 5→3′and 3→5′ exonuclease activity, enzyme cofactors such as magnesium ormanganese, salts, chain extension nucleotides such as deoxynucleosidetriphosphates (dNTPs) or biotinylated dNTPs, necessary to carry out anassay or reaction, such as amplification and/or detection of methylatedtarget nucleic acid sequences in the MLH1 promoter.

In one embodiment, the kits of the present technology further comprisepositive control methylated DNA sequences and negative controlunmethylated DNA sequences to correct for any amplification variabilitybetween samples. A kit may further contain a means for determining theextent of methylation within the MLH1 promoter, and a means forcomparing the extent of methylation with a standard. The kit may alsocomprise instructions for use, software for automated analysis,containers, packages such as packaging intended for commercial sale andthe like.

The kit may further comprise one or more of: wash buffers and/orreagents, hybridization buffers and/or reagents, labeling buffers and/orreagents, and detection means. The buffers and/or reagents are usuallyoptimized for the particular amplification/detection technique for whichthe kit is intended. Protocols for using these buffers and reagents forperforming different steps of the procedure may also be included in thekit.

The kits of the present technology may include components that are usedto prepare nucleic acids from a test sample for the subsequentamplification and/or detection of methylated target nucleic acidsequences in the MLH1 promoter. Such sample preparation components canbe used to produce nucleic acid extracts from any bodily fluids (such asblood, serum, plasma, etc.) or from tissue samples. The test samplesused in the above-described methods will vary based on factors such asthe assay format, nature of the detection method, and the specifictissues, cells or extracts used as the test sample to be assayed.Methods of extracting nucleic acids from samples are well known in theart and can be readily adapted to obtain a sample that is compatiblewith the system utilized. Automated sample preparation systems forextracting nucleic acids from a test sample are commercially available,e.g., Roche Molecular Systems' COBAS AmpliPrep System, Qiagen's BioRobot9600, and Applied Biosystems' PRISM™ 6700 sample preparation system.

EXAMPLES Example 1: MLH1 Methylation Assay Design

FIG. 1 shows the nucleic acid sequences of the MLH1 promoter ‘C’ region,MLH1 intron 14, and ACTB amplicons, as well as the corresponding probeand primer sequences for each amplicon. The primers and probes of allthree of the amplicons were designed to have very similar T_(m)characteristics allowing them to function with similar efficiencies inthe multiplex PCR reactions. Experiments that tested the efficiency ofthe primer pairs for each amplicon showed similarly high efficienciesfor each amplicon (e.g., ˜85-110%, usually around 95%), regardless ofwhether the PCR reaction was run singly or as a multiplex.

As shown in FIG. 1, the nucleic acid sequence of the MLH1 promoter ‘C’region amplicon contains 2 recognition sites for themethylation-sensitive restrictive endonuclease HhaI, whereas the nucleicacid sequence of the MLH1 intron 14 amplicon contains 2 recognitionsites for the methylation-insensitive restrictive endonuclease AluI. Thetarget nucleic acid sequence at intron 14 of MLH1 was specificallyselected as the internal control for enzymatic digestion in each testsample because of the enzymatic compatibility between the HhaI and AluIrestriction enzymes. Both HhaI and AluI have 100% activity in CutSmartbuffer, have an optimal incubation temperature of 37° C., and can beheat-inactivated at 95° C.

Additionally, no SNPs or COSMIC catalogued mutations were identified ineither the MLH1 promoter ‘C’ region amplicon or the MLH1 intron 14amplicon using UCSC Genome Browser, thereby eliminating a potential riskfor false positive results.

The impact of high salt concentration of the restriction enzyme digestson the integrity of the MLH1 methylation assay of the present technologywas also examined. Experiments that used QuantiTect MMX (Qiagen) bufferfor the multiplex PCR reaction for a given sample (methylated orunmethylated) yielded Ct values that were similar to the Ct valuesobtained when the same sample was incubated with QuantiTect MMX (Qiagen)buffer and 1× CutSmart buffer. These results demonstrate that anydifferences observed when using higher salt concentrations areinconsequential and do not disrupt the ability of the assay todiscriminate between methylated and unmethylated samples.

Example 2: Detection of Methylation of MLH1 Promoter ‘C’ Region in FFPEand Whole Blood Samples

This Example demonstrates that the MLH1 methylation assay of the presenttechnology can effectively discriminate between methylated andunmethylated samples, including FFPE tissues.

Genomic DNA was extracted from FFPE tissue or whole blood samples fromCRC patients using standard protocols. The extracted DNA was quantifiedusing the NanoDrop ND-1000 and dilutions for each sample were preparedaccording to the following guidelines:

Diluted Input DNA/Well NanoDrop Prepare Concen- (ng/well = 2 μL × QuantDilution tration diluted conc.) (ng/μL) Dilution (need ≥5 μL): (ng/μL)Target = 50-100 ng  1-49 None N/A  1-49  2-98*  50-100 1:2 3 μL DNA +25-50 50-100 3 μL H₂O 101-200 1:4 2 μL DNA + 25-50 50-100 6 μL H₂O201-250 1:5 1 μL DNA + 40-50 80-100 4 μL H₂O 251-500  1:10 1 μL DNA +25-50 50-100 9 μL H₂O  501-1000  1:20 1 μL DNA + 25-50 50-100 19 μL H₂O

The “no enzyme” master mix was prepared as follows:

Final Concentration x1 Rxn (μL) x50 Rxn (μL) CutSmart Buffer (10X) 1X 150 H₂O (QuantiTect kit) — 7 350 Total master mix: 8 400 +DNA 50 ng-100ng 2 Total reaction: 10

The HhaI-AluI dual digest master mix was prepared as follows:

Final Concentration x1 Rxn (μL) x50 Rxn (μL) CutSmart Buffer (10X) 1X 150 HhaI enzyme (20 U/μL) 5 U 0.25 12.5 AluI enzyme (10 U/μL) 5 U 0.5 25H₂O (QuantiTect kit) — 6.25 312.5 Total master mix: 8 400 +DNA 50 ng-100ng 2 Total reaction: 10

After preparing a “no enzyme” reaction and HhaI-AluI dual digestreaction for each sample in a 96-well PCR plate, the samples wereincubated at 37° C. for 16 hours. The restriction enzymes weresubsequently heat-inactivated at 95° C. for 20 minutes.

The master mix for the MLH1 multiplex PCR assay was prepared in a deadair box as follows:

MLH1 Multiplex Master Mix Preparation x1 Rxn (μL) x28 Rxn (μL) x100 Rxn(μL) Final Concentration QuantiTect 2X MMX 10 280 1000 1X Buffer MLH1promoter ‘C’ region 0.1 2.8 10 Forward primer 200 nM Primer/Probe MixReverse primer 200 nM Probe 100 nM MLH1 intron 14 0.13 3.64 13 Forwardprimer 200 nM Primer/Probe Mix Reverse primer 200 nM Probe 250 nM ACTBPrimer/Probe Mix 0.13 3.64 13 Forward primer 200 nM Reverse primer 200nM Probe 250 nM Master Mix Total 10.36 290.08 1036

10.3 μL of prepared MLH1 multiplex master mix was added to each well ofa LifeTech MicroAmp Fast Optical 96-well PCR plate. Followingrestriction digestion, the DNA samples were directly added to the wellscontaining the multiplex PCR master mix. The PCR plate was sealed withMicroAmp Optical Adhesive Film and transferred to the Viia™ 7 real-timePCR instrument.

The ultimate real-time quantitative PCR protocol employed is shownbelow:

Enzyme Activation  95°-20 min 1 cycle PCR 95°-15 sec 40 cycles 62°-30sec 72°-30 sec Cooling 40°-10 sec 1 cycle

The real-time quantitative PCR data was analyzed using the Viia™ 7software. MeScore calculations were performed using the formulasdescribed herein.

A MeScore>0.285 is considered positive for MLH1 promoter methylation,whereas a MeScore≤0.285 is considered negative for MLH1 promotermethylation. The MeScore cut-off (which was calculated using the MedCalcSoftware) is based on the results of a validation study involving 104samples that were tested using both the MLH1 methylation assay of thepresent technology, and a bisulfite conversion assay. FIG. 3 shows theROC curve of the MLH1 methylation assay of the present technology, whichwas generated with the results of the bisulfite conversion validationstudy shown in FIGS. 4A and 4B. The predicted sensitivity andspecificity of the MLH1 methylation assay of the present technology was94.1% and 96.6% respectively at a threshold MeScore of >0.285.

Results.

As shown in FIG. 2B, incubation with the methylation-insensitive enzymeAluI, which cleaves both unmethylated and methylated DNA, resulted inlarger Ct values for the MLH1 intron 14 amplicon compared to the noenzyme control samples. In contrast, FIG. 2C shows that the Ct valuesfor the ACTB amplicon of the dual digested samples were similar to theCt values observed in the no enzyme control samples. FIGS. 2A and 2Bshow that the baseline enzymatic activity of HhaI on an unmethylated DNAcontrol sample is comparable to that observed with AluI, as measured bythe deltaCt between the no enzyme control and the digested samples(deltaCt=8.5 vs. 6.0 respectively). FIG. 2A also shows that the deltaCtvalues of the MSI+ FFPE samples were comparable to that observed in themethylated DNA control sample, thus demonstrating that the MLH1 promoterin these MSI+ samples was methylated.

The computed MeScore for each of the representative samples shown inFIGS. 2A-2C are shown below in Table 1. The observed variations inMeScore in the BRAF V600E+ and MSI+ CRC samples can be attributed to theheterogeneous nature of CRC FFPE samples, which may be due to partialmethylation of the MLH1 promoter. The accuracy of the MeScore for thetested samples was independently confirmed via bisulfite conversion. Asshown in Table 1, the overall concordance of the MLH1 MS-MLPA assay ofthe present technology (when compared to the bisulfite conversionresults) is 93.75%.

TABLE 1 MLH1 Ratio C Ratio promoter MLH1 1 - (MLH1 ACTB ‘C’ intronpromoter C 1 - (|ACTB MeScore region 14 ACTB dCt/MLH1 dCt|/MLH1 RatioC/Ratio Bisulfite Sample Group Samples dCt dCt dCt intron 14 dCt) intron14 dCt) ACTB Result Control Negative UnMeDNA 8.5 6.0 −0.4 −0.45 0.92−0.46 neg control (100 ng) Positive control MeDNA 0.1 5.1 −0.1 0.98 0.971.01 POS (100 ng) Possible MSI+ (FFPE) 18121T 0.019 1.8 0.0 0.99 0.990.99 POS Methylation- MSI+ (FFPE) 139T −0.8 1.8 −1.2 1.47 0.32 4.56 POSPositive FFPE BRAF V600E+ 12646 2.1 5.8 0.5 0.63 0.92 0.68 POS (FFPE)MSI+ (FFPE) 17191T −0.5 1.1 −0.5 1.47 0.60 2.44 POS BRAF V600E+ 132253.0 4.1 0.6 0.26 0.86 0.298 POS (FFPE) Likely Methylation- Matched “N”of 18122N 2.3 3.3 0.2 0.29 0.93 0.32 neg Negative FFPE or WB MSI-neg(FFPE) BRAF V600E− 14634T 1.9 2.4 −0.2 0.19 0.91 0.21 neg neg (FFPE)Matched “N” of 15844N 3.7 4.0 −0.2 0.08 0.95 0.08 neg BRAF V600E+ (FFPE)BRAF V600E− 17544 4.9 3.1 0.2 −0.59 0.95 −0.63 neg neg (FFPE) BRAFV600E− 17213T 5.4 3.8 −0.4 −0.42 0.89 −0.47 neg neg (FFPE) BRAF V600E−16997 2.7 2.3 −0.1 −0.16 0.96 −0.17 neg neg (FFPE) BRAF V600E− 16489 1.81.6 0.4 −0.11 0.75 −0.15 neg neg (FFPE) Matched whole 17191WB 12.0 9.3−0.6 −0.297 0.94 −0.32 neg blood of MSI-neg Whole blood 18671WB 9.1 9.7−0.6 0.06 0.94 0.06 neg from JAK validation Concordance (provisionalMeScore cutoff of POS > 0.285; see FIG. 4A) 15/16 concordant = 93.75%

Further, Table 2 demonstrates that the overall specificity for samplegroups expected to be negative for MLH1 promoter methylation is 93%.

TABLE 2 MeScore Specificity Sample Group algorithm- for expected (allexpected to be negative for MLH1 negative negative promoter methylation)N (≤0.285)* results MSI+ matched “Normal” FFPE tissue 11  9/11  82%MSI-neg FFPE tumor tissue 12 12/12 100% MSI-neg matched “Normal” FFPEtissue 9 8/9  89% BRAF V600E-neg matched “Normal” 1 1/1 100% FFPE tissueWhole blood 9 9/9 100% Overall negative concordance 39/42  93%

These results demonstrate that the MLH1 MS-MLPA assay of the presenttechnology can detect methylation of the MLH1 promoter in FFPE tissuesamples with high specificity and sensitivity. Therefore, DNAdegradation and partial DNA denaturation during embedding of the tissuesdo not appear to influence the accuracy of the results of the MLH1methylation assay of the present technology.

Accordingly, these results demonstrate that the MLH1 MS-MLPA assay ofthe present technology is useful for detecting aberrant methylation ofthe MLH1 promoter ‘C’ region in a sample. Further, these resultsdemonstrate that the MLH1 MS-MLPA assay of the present technology isuseful in methods for excluding Lynch syndrome as a possible diagnosisin a CRC or endometrial cancer patient.

EQUIVALENTS

The present technology is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the present technology. Manymodifications and variations of this present technology can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. Functionally equivalent methods andapparatuses within the scope of the present technology, in addition tothose enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the present technology. It is to beunderstood that this present technology is not limited to particularmethods, reagents, compounds compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

The invention claimed is:
 1. A method for detecting methylation of atarget nucleic acid sequence in the promoter of MLH1 in a samplecomprising (a) incubating the sample comprising double-stranded genomicDNA with at least one methylation-sensitive restriction enzyme and atleast one methylation-insensitive restriction enzyme, wherein (i) themethylation-sensitive restriction enzyme and the methylation-insensitiverestriction enzyme are not isoschizomers of each other; (ii) themethylation-sensitive restriction enzyme cleaves the double-strandedgenomic DNA at unmethylated recognition sites for themethylation-sensitive restriction enzyme, leaving methylated recognitionsites for the methylation-sensitive restriction enzyme intact; (iii) themethylation-insensitive restriction enzyme cleaves the double-strandedgenomic DNA at both methylated and unmethylated recognition sites forthe methylation-insensitive restriction enzyme; (iv) the target nucleicacid sequence in the promoter of MLH1 in the sample comprises arecognition site for the methylation-sensitive restriction enzyme; and(v) a target nucleic acid sequence at intron 14 of MLH1 in the samplecomprises a recognition site for the methylation-insensitive restrictionenzyme; (b) incubating the sample with a plurality of probes forquerying a plurality of target nucleic acids in the sample, wherein theplurality of probes comprises (i) a first locus specific probecomprising a first target specific region complementary to the targetnucleic acid sequence in the promoter of MLH1, wherein the sequence ofthe first locus specific probe is 5′ AAGCACCTCCTCCGCTCTGC 3′ (SEQ IDNO: 1) or a complement thereof and wherein the first locus specificprobe is a single oligonucleotide; and (ii) a second locus specificprobe comprising a second target specific region complementary to thetarget nucleic acid sequence at intron 14 of MLH1, wherein the secondlocus specific probe is a single oligonucleotide and wherein the firstlocus specific probe and second locus specific probe are detectablylabelled; (c) hybridizing the plurality of probes to the plurality oftarget nucleic acids in the sample to form a plurality of hybridizationcomplexes; (d) amplifying the plurality of hybridization complexes viareal-time quantitative PCR to produce a plurality of amplicons, whereinamplification is carried out with a plurality of primer sets comprising(i) a first forward primer comprising a region that is complementary toa nucleic acid sequence located 5′ from the target nucleic acid sequencein the promoter of MLH1; (ii) a first reverse primer comprising a regionthat is complementary to a nucleic acid sequence located 3′ from thetarget nucleic acid sequence in the promoter of MLH1; (iii) a secondforward primer comprising a region that is complementary to a nucleicacid sequence located 5′ from the target nucleic acid sequence at intron14 of MLH1; and (iv) a second reverse primer comprising a region that iscomplementary to a nucleic acid sequence located 3′ from the targetnucleic acid sequence at intron 14 of MLH1; and (e) detecting theplurality of amplicons, wherein detecting an amplicon comprising thetarget nucleic acid sequence in the promoter of MLH1 indicatesmethylation of the target nucleic acid sequence in the promoter of MLH1in the sample, wherein the sample is obtained from a formalin fixedparaffin-embedded tissue sample, and wherein the methylation-sensitiverestriction enzyme is HhaI.
 2. The method of claim 1, wherein theplurality of probes further comprises a third locus specific probecomprising a third target specific region complementary to a targetnucleic acid sequence at ACTB, wherein the target nucleic acid sequenceat ACTB does not contain a recognition site for themethylation-sensitive restriction enzyme and the methylation-insensitiverestriction enzyme; and wherein the third locus specific probe is asingle oligonucleotide and is detectably labelled.
 3. The method ofclaim 2, wherein the first locus specific probe, the second locusspecific probe and the third locus specific probe are detectablylabelled with distinct fluorophores.
 4. The method of claim 3, whereinthe fluorophores are selected from the group consisting of FAM, CY5 andHEX.
 5. The method of claim 2, wherein the third locus specific probecomprises the sequence of 5′ TGAACCTGTGTCTGCCACTGTGTG 3′ (SEQ ID NO: 3)or a complement thereof.
 6. The method of claim 2, wherein the pluralityof primer sets further comprises a third forward primer comprising aregion that is complementary to a nucleic acid sequence located 5′ fromthe target nucleic acid sequence at ACTB; and a third reverse primercomprising a region that is complementary to a nucleic acid sequencelocated 3′ from the target nucleic acid sequence at ACTB.
 7. The methodof claim 6, wherein the third forward primer comprises the sequence of5′ GGCTCAGCAAGTCTTCTGG 3′ (SEQ ID NO: 8) and the third reverse primercomprises the sequence of 5′ CCTGGTGGGAAAGATGACC 3′ (SEQ ID NO: 9). 8.The method of claim 1, wherein the second locus specific probe comprisesthe sequence of 5′ CTACAACAATGGTCCAGGGAGCACA 3′ (SEQ ID NO: 2) or acomplement thereof.
 9. The method of claim 1, wherein the first forwardprimer comprises the sequence of 5′ AGGAGGAGCCTGAGAAGC 3′ (SEQ ID NO: 4)and the first reverse primer comprises the sequence of 5′CTTGTGTGCCTCTGCTGAG 3′ (SEQ ID NO: 5).
 10. The method of claim 1,wherein the second forward primer comprises the sequence of 5′CTGAGTGTGTGAACAAGCAGAG 3′ (SEQ ID NO: 6) and the second reverse primercomprises the sequence of 5′ ACCTCATGCTGCTCTCCTTAG 3′ (SEQ ID NO: 7).11. The method of claim 1, wherein the methylation-insensitiverestriction enzyme is AluI.
 12. The method of claim 1, wherein thetarget nucleic acid sequence in the promoter of MLH1 corresponds to theMLH1 promoter ‘C’ region.
 13. The method of claim 1, wherein the sampleis derived from a subject diagnosed with colorectal or endometrialcancer.
 14. The method of claim 13, wherein the subject diagnosed withcolorectal or endometrial cancer is positive for BRAF V600E ormicrosatellite instability (MSI).
 15. The method of claim 1, wherein thesample is derived from a subject suspected of having Lynch syndrome. 16.The method of claim 15, wherein the subject suspected of having Lynchsyndrome displays tumors in one or more of colon, rectum, endometrium,stomach, ovary, urinary tract, and small intestine.
 17. The method ofclaim 16, wherein the tumors of the subject suspected of having Lynchsyndrome show a loss of MLH1 protein expression usingimmunohistochemical (IHC) methods.
 18. The method of claim 16, whereinthe tumors of the subject suspected of having Lynch syndrome arepositive for MSI.